Methods and apparatus for carrier-free deflection electrophoresis

ABSTRACT

The invention relates to a method and apparatus for carrier-free deflection electrophoresis, in which a separating media and a sample to be examined flow through a separating chamber between a pair of electrodes in a series of reversing bulk fluid flow along the direction of the electrodes, thereby separating the sample into zones which are to be collected into fractions for analysis or further processing. Among other things, the apparatus and method enable high-resolution separation of particles that can be performed in miniaturized chambers in electrophoresis modes including isoelectric focusing, zone electrophoresis, and isotachophoresis.

This patent application claims priority to U.S. Provisional PatentApplication Ser. No. 60/823,833 filed on Aug. 29, 2006 and U.S.Provisional Patent Application Ser. No. 60/883,260 filed on Jan. 3,2007.

FIELD OF THE INVENTION

The invention relates to carrier-free deflection electrophoresis,including an apparatus and method for carrying out the same.

BACKGROUND OF THE INVENTION

The basics of carrier-free deflection electrophoresis, especially withrespect to continuously operated processes, have been described in theliterature over 30 years ago. The process is sometimes described underthe term FFE (free flow electrophoresis) or more commonly CFE(continuous flow electrophoresis). (K. Hannig: Carrier-free continuouselectrophoresis and its application. Anal. Chem. 181, 233 (1961); M. C.Roman and P. R. Brown: Anal. Chem. Free Flow Electrophoresis. 66(N2),86-94, (1994); R. Braun, H. Wagner and G. Weber: Preparative Free FlowElectrophoresis—a powerful procedure for separating natural substances,GIT Fachzeitschrift für das Laboratorium 39 (1995), 317-322).

Generally, FFE separation procedures are used to separate ions of anymolecular weight up to bioparticles. It is here irrelevant whether thesample to be separated is charged itself, or whether the charge cameabout via the addition or sorption of ions.

Carrier-free deflection electrophoresis has traditionally been used incontinuous processes commonly referred to as continuous free-flowelectrophoresis (CFFE). This method used in absence of a support matrixsuch as a gel enables the separation, fractionation, and possibleisolation of both soluble and insoluble components. In comparison toother methods enabling isolation of separated sample components,continuous free-flow electrophoresis generally offers three mainadvantages: (i) the sample is maintained in a liquid medium/in solutionwhich can be directly used for further processing, (ii) the separationmay be performed continuously and enables one to obtain as much ashundreds of milligrams or even gram amounts of pure substances per hourand (iii) the separation is gentle and preserves biological activity ofthe separated components.

The technology of FFE is particularly useful in the separation andfractionation of complex proteins, and is thus applicable to theemerging field of proteomics, which is growing increasingly important inacademic and pharmaceutical research as well as the generalbiotechnology and clinical diagnostic markets. For example, as proteomicresearch has grown, there has been an increased demand in theimprovement of protein separation performance or resolution, especiallyin terms of resolution process reliability. There has also been demandfor a universal front-end separation system and method that occurs priorto a later analysis or further separation/fractionation step.

Improvements to the field of FFE have come about. For example, theprocess of continuous deflection electrophoresis (or continuousfree-flow electrophoresis) has been improved by way of stabilizationmedia and counter-flow media. This is reflected, for example, in U.S.Pat. No. 5,275,706, the disclosure of which is hereby incorporated byreference in its entirety. According to this patent, a counter-flowmedium is introduced into the separation space counter to the continuousflow direction of the bulk separation medium and sample that travelsbetween the electrodes. Both media (separation media and counterflowmedia) are discharged or eluted through fractionation outlets typicallyinto a microtiter plate, resulting in a fractionation process having alow void volume. Additionally, a laminar flow of the media in the regionof the fractionation outlets is maintained (i.e., with very low or noturbulence).

Additionally, free-flow deflection electrophoresis has been implementedin a non-continuous or interval process. For example, a process ofnon-continuous deflection electrophoresis is shown in U.S. Pat. No.6,328,868, the disclosure of which is hereby incorporated by reference.In this patent, the sample and separation medium are both introducedinto an electrophoresis chamber, and then separated using anelectrophoresis mode such as zone electrophoresis, isotachophoresis, orisoelectric focusing, and are finally expelled from the chamber throughfractionation outlets. Embodiments of the '868 patent describe theseparation media and sample movement to be unidirectional, travelingfrom the inlet end towards the outlet end of the chamber, with aneffective voltage applied causing electrophoretic migration to occurwhile the sample and media are not being fluidically driven from theinlet end towards the outlet end. Examples of embodiments of the '868patent are shown in FIG. 1.

Both above examples of FFE (i.e., continuous and non-continuous orinterval mode) can be used in certain situations, with each experimentalgoal having factors and requirements or specifics that lend one toprefer one process to the other. Such factors include the choice ofsample intended to be separated including required or desired separationtime, sample size, separation resolution desired, chamber size, etc.These and other factors influence the mode of separation as well as theapparatus, specific methods, techniques, and compositions to be used.One or more of these above factors may or may not influence which modeof operation (continuous or non-continuous) is chosen when both areavailable to the user given a certain situation and experimental orseparation goal. It should be noted that while using free-flowelectrophoresis, both in continuous and non-continuous (or interval)modes of operation, each may have many benefits when compared to otherseparation or fractionation methodologies and techniques. Nevertheless,improvements are always desired.

Numerous publications describe the physical or electrochemical effectsthat contribute to the so-called “band widening” of the analytes duringseparation in continuous free-flow deflection electrophoresis (J. A.Giannovario, R. Griffin, E. L. Gray: A mathematical model of free-flowelectrophoresis. Journal of Chromatography, 153, 329-352 (1978); F. G.Boese: Contribution to a mathematical theory of free flowelectrophoresis, J. Chromat. 483, 145-170 (1988); K. Hannig and H. G.Heidrich: Free-Flow Electrophoresis, 1990 by GIT Verlag Darmstadt ISBN3-921956-88-9).

The most important of these effects inherent in continuous FFE are:

1. band widening due to the laminar flow profile;

2. band widening due to thermal convection;

3. band widening due to electrical osmosis;

4. band widening due to electrokinetic effects.

The negative influence of all electrokinetic effects described thus farcan be minimized or eliminated by using separation media with suitableionic constituents with sufficiently high ionic strength, and at thesame time not excessively increasing the concentration of the sample.

There are numerous ways to minimize the negative influence of electricalosmosis, e.g., through the selection of a suitable wall material(plastics instead of glass or quartz), or most preferably by addingsurface-active chemicals to the separation media that precludeelectrical osmosis. This method is referred to as “dynamic coating” inthe literature.

The negative influence of thermal convection can be reduced very easilyby arranging and operating the electrophoresis chamber horizontallyinstead of vertically. Additionally, thermal effects can be minimized byappropriate cooling and maintaining the electrophoresis chamber at aconstant temperature throughout the separation process.

The negative influence of the laminar flow profile is not observed forcontinuous isoelectric focusing (IEF) as long as a sufficiently longseparation time is selected that also enables the focusing of theanalytes, which are transported at the highest linear velocity in thecenter of the electrophoresis chamber gap.

By contrast, the negative influence is very significant in the case ofthe electromigration processes. Analytes that migrate near or in theboundary surface to the walls of the electrophoresis chamber passthrough the electrophoresis chamber in a considerably longer time thananalytes at the center of the electrophoresis chamber gap, and aretherefore deflected to a clearly greater extent due to their longerresidence time. This effect results in a band widening detectable as atailing in the direction of electromigration.

Given a continuously executed electromigration process under theboundary conditions of carrier-free electrophoresis, the negativeinfluence of the laminar flow profile cannot be averted forlow-molecular analytes. The absolute value of band widening increases asdoes the migration distance of the analytes. Reducing the diffusionrates for analytes that have their migration impacted by usingseparation media with increased viscosity also does not help, since thismagnifies the unfavorable nature of the laminar flow profile.

In the case of separation of bioparticles, a quantitatively reducedsample feeding to the center of the electrophoresis chamber gap canresult in an improved resolution, since the particles cannot get intothe area of the electrophoresis chamber walls during a retention time of<10 minutes due to the extremely low diffusion. However, the influenceof laminar flow profile can only be minimized in this way by distinctlyreducing the sample feeding rate (e.g., to a sample flow rate that isonly 0.1% to 0.5% of the flow rate of the separation medium).

Compared to the continuous FFE operating modes known in the art, theinterval FFE mode as described in U.S. Pat. No. 6,328,868 is capable ofavoiding the negative influence of a laminar flow profile observed inelectromigration processes.

However, there remains a need in the art for further improvements of thefree-flow electrophoresis methods.

SUMMARY OF THE INVENTION

Accordingly, the object of the invention is to provide a method forcarrier-free deflection electrophoresis, which eliminates the influenceof a laminar flow profile typical of continuous free-flowelectrophoretic separations and additionally increases separationquality and/or reducing the turn around time or reduction infractionation time needed to obtain high resolution quality forelectrophoretic separations. The method and device of the presentinvention can be used for both preparative and analytical separations.

Accordingly, in a first aspect, the present invention relates to amethod for separating particles, wherein the method comprises disposinga separation medium and sample in an electrophoresis chamber having atop plate, a bottom plate and a plurality of electrodes generallyparallel to one another with a separation space disposed therebetween,and a fluidic displacement system for conveying separation medium andsample particles between the electrodes, applying a voltage between theelectrodes effective to manipulate particles electrophoretically, andwherein at least a portion of the separation medium and sample isdisplaced towards a first direction generally parallel to the directionof the electrodes, and subsequently in a second direction generallyopposite the first direction.

In certain embodiments, the method for separating particles comprisesdisposing a separation medium and sample in an electrophoresis chamberhaving a top plate, a bottom plate, a first chamber end, a secondchamber end, and a plurality of electrodes generally parallel to oneanother with a separation space disposed therebetween, the electrodeslongitudinally extending toward each of the ends, and a fluidicdisplacement system for conveying a separation medium between the firstand second chamber ends, applying a voltage between the electrodeseffective to manipulate particles electrophoretically, displacing atleast a portion of the separation medium and sample towards the firstchamber end, displacing at least a portion of the separation medium andsample towards the second chamber end (i.e. reversal of the bulk flow),and optionally displacing at least a portion of the separation mediumand sample towards the first chamber end, for example so as to dispel orelute at least a portion of the sample and/or separation medium throughone or more outlets that are located at the chamber end opposite to theinlets. “Generally parallel” in the context of the present inventionmeans that the bulk flow direction of at least the non-charged particlesin the separation medium (e.g. water) is essentially parallel to theelongated electrodes. However, those of skill in the art will appreciatethat charged species (i.e., sample or ions in the separation medium)within the electrophoresis chamber may at the same time also bedeflected by the electrical field between the electrodes, thereby movingtowards the cathode or anode and at the same time parallel to theelectrodes towards the inlet or outlet end of the electrophoresischamber. The movement of particles during a separation within a FFEapparatus is described in more detail further down below.

The present invention offers a distinct improvement in the quality ofthe electrophoretic separation, and in long-term stability and improvedspeed to achieve high resolution for the electrophoretic separation whencompared to other separation techniques. In addition, the method of thepresent invention also offers increased flexibility regarding the designof the FFE devices used for the electrophoretic separations. Embodimentsdisclosed herein can be applied to most separation protocols thattypically have been performed using continuous or static interval freeflow electrophoresis applications generally known in the art anddescribed hereinabove, including zone electrophoresis, isoelectricfocusing and isotachophoresis.

Typically, chemical and physical discontinuities exist inelectrophoretic chambers across the separation area and are well knownsources of turbulences for liquids in the areas adjacent the migratingboundaries of the media, whereas non migrating boundaries (likeboundaries between stabilizing media and separation media) will giverise to turbulences in case of overloading the separation process only(due to high field strength or to high current). It has now been foundthat the turbulences at migrating boundaries can be reducedsubstantially by shear forces, given by the vector of a linear movementof the separation media forward and backwards inside the separation cellwith respect to the generally fixed electrophoresis chamber walls. Thecycling back and forth, forward and backwards inside the separation cellwith respect to generally fixed electrophoresis chamber walls is hereintermed cyclic free-flow electrophoresis or, alternatively, cyclicinterval free-flow electrophoresis.

For the purposes of the present invention, cyclic FFE includes at leastone full cycle (i.e., moving separation medium and sample away from thesample and media inlets towards the opposite end of the FFE apparatus,then moving the sample and separation media in a second (typicallyopposite) direction, i.e., towards the inlet end of the FFE apparatus.In addition to the cycles during which electrophoretic separation of thesample particles is achieved, cyclic FFE may further comprise a stagewherein the direction is changed again towards the outlet end of the FFEapparatus in order to elute the sample and bulk separation media.Similarly, when the outlets are placed at or near the inlets of theapparatus, one full cycle is characterized by a single reversion of theflow direction, i.e. moving the separation medium and sample towards afirst direction of the FFE apparatus, then moving the sample andseparation media in a second (typically opposite) direction towards theinlet, and in this instance also outlet end of the FFE apparatus. Thecycle described above may be repeated multiple times as furtherdescribed hereinbelow, or may be carried out only once. Preferably, themethod is supplemented by dispelling/eluting the sample and bulkseparation media through one or more outlets.

In preferred embodiments, the number of cycles is greater than one, suchas at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles, and is selected so asto achieve sufficient separation of the sample before the sample or atleast fractions thereof is/are collected at the outlet end of the FFEapparatus. It will be understood by the skilled person that the numberof cycles displacing the sample and the bulk separation medium back andforth depends on a number of factors, including sample size, quality ofseparation, electrophoretic mobility of the sample among others.

Electrophoretic mobility (EM), as used herein, means the rate ofmigration of anions and cations in an electrical field at a given fieldstrength per time unit in an aqueous media. The electrophoretic mobilityu can be calculated as follows:

u=s/H×t

wherein s represents the distance of migration (m), H represents theelectric field strength (V/m) and t represents the time (sec.).

An improved quality of separation is achieved using the novel method andapparatus according to embodiments of the present invention incombination with any of the known separation modes of free-flowelectrophoresis, e.g., isotachophoresis (ITP), zone electrophoresis(ZE), and isoelectric focusing (IEF). Specifically, experiments haveshown that the benefits of cyclic interval FFE are most valuable andsurprising in the cases of cyclic FF-isotachophoresis and of cyclicFF-zone electrophoresis (for ZE, see FIGS. 5 and 6).

As mentioned above, U.S. Pat. No. 6,328,868 to Weber describes a methodfor carrying out matrix-free or free-solution electrophoresis betweenelectrodes in a general interval manner. The interval mode described inthe '868 invention generally introduces and elutes the sample andseparation media in one direction. This direction, unlike traditional(gel and matrix-free) capillary electrophoresis wherein the sample movesin the same direction as the electric field through the capillary, isshared by the orientation of the elongated electrodes. An example offive stages (stages 0 through stage 4) demonstrating one exemplaryembodiment of the '868 invention is shown in FIG. 1, as represented byFIGS. 1 a-1 e respectively. As shown in FIG. 1 a, stage 0 comprisesintroducing a sample and separation media into the electrophoresischamber with the flow on and the voltage off. As shown in FIG. 1 b,stage 1 maintains the voltage off and the bulk flow of sample andseparation media is stopped (flow off). As shown in FIG. 1 c, stage 2maintains the bulk flow off, but applies voltage between the electrodescausing electrophoretic separation. As shown in FIG. 1 d, stage 3 turnsthe voltage off but forces the bulk flow of sample and separation mediatowards outlets opposite the inlets where the separation media wasintroduced. Finally, as shown in FIG. 1 e, stage 4 turns the bulk fluidflow and voltage off. Stages 0-4 can then be repeated if so desired.

In the case of the '868 invention, displacement or acceleration of thesample caused by a pump or some other fluidic displacement element inthe electrophoresis chamber between the electrodes only takes place whenthe voltage is off or at least when the voltage is ineffective forelectrophoretic migration, i.e., when no part of the sample is beingsubjected to an effective electrophoretic field strength. Additionally,absolute displacement of the bulk sample and media in the '868 inventiononly occurs in the direction starting from the sample inlets and endingat the sample outlets. These two characteristics as well as otherelements of the method and apparatus of the '868 invention were improvedupon in the present invention.

Many advantages of the new FFE-process in cyclic interval mode accordingto embodiments of the present invention have been observed through anumber of experiments.

First, because the sample and separation media is in motion during theelectrophoretic separation stage, it is possible to apply higher fieldstrengths. In view of the higher field strengths the new cyclic intervalFFE-process will provide an enhanced performance, which can be used fora better discrimination of the analytes given the same duration ofprocessing time that would be typical of the method described in U.S.Pat. No. 6,328,868 to Weber. Alternatively, the new method offers equaldiscrimination quality of the analytes but can be performed at a reducedduration of processing time when compared to continuous or standardinterval free-flow electrophoresis methods. The latter advantage couldenable higher throughput of distinct separations which may be applicableto an electrophoresis process used, e.g., in clinical proteomics.

Second, all conventional continuous FFE separation techniques andprotocols may be easily adopted for use in the cyclic interval free-flowelectrophoresis process.

Third, the method and apparatus according to embodiments of the presentinvention allows to extend the duration of electrophoretic separation ofa sample in a chamber due to the periodic reversal of bulk sample flow.For instance, a continuous free-flow electrophoresis machine with aminimum bulk sample and separation media flow rate, controlled by apump, has a minimum duration of travel that is a function of theviscosity, pump flow rate, shape of the chamber, and length from inletto outlet. Unlike the continuous free-flow electrophoresis method, thecyclic interval method according to embodiments of the presentinvention, with the same minimum flow rate, viscosity, shape of chamber,and ability to reverse the fluid flow, can prolong the duration oftravel between the sample inlets and outlets. This provides anopportunity for a prolonged time of separation which, for example, inthe case of FF-isoelectric focusing is especially powerful and useful.Since the sample in the cyclic interval mode can be moved repeatedly ina forward and reverse direction, the effective travel length of thesample between the electrodes can be extended indefinitely. Theprolonged time or extended distance of travel for separated particleswill result in a better focusing of analytes, especially for those witha relatively low electrophoretic mobility.

Fourth, the new cyclic interval free-flow electrophoresis process can beutilized in separation chambers with many different options of geometricconfiguration and cross-section. This therefore enables flexibility forhow one may implement FFE in miniaturized electrophoresis chambers, theshapes and sizes of which can be altered depending on the desired numberof sample outlets for elution interposed between the pair or pairs ofelectrodes. Additionally, the length of the electrodes can be controlledor designed such that the amount of sample and separation media can beadjusted to account for the sample size or sample and analyteconcentration that needs to be fractionated and/or detected.

The new cyclic interval FFE mode according to embodiments of the presentinvention can be used for specific applications hitherto inaccessible tofree-flow electrophoretic separation technology. For example, theseparation and isolation of protein isoforms with rather low speed ofelectrophoretic migration was difficult to achieve. With the methoddescribed herein, isolation of, e.g., such isoforms can be accomplishedusing the cyclic interval free-flow electrophoresis mode disclosedherein, which historically could not be realized using the continuousFFE-processes existing in the art. In addition, many other separationproblems employing a variety of different samples have been successfullyaccomplished using the new cyclic interval FFE mode of the presentinvention.

The description below as well as the enclosed Figures exemplify severalembodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 describes steps for carrying out interval free-flowelectrophoresis of the prior art.

FIG. 2 describes steps for carrying out an embodiment of the presentinvention.

FIG. 3 describes an FFE apparatus suitable for carrying out free-flowelectrophoresis according to an embodiment of the present invention.

FIGS. 4 a and 4 b describe another setup embodiment suitable forcarrying out free-flow electrophoresis according to an embodiment of thepresent invention.

FIG. 5 demonstrates performance results of the prior art continuous FFE.

FIG. 6 demonstrates performance results of the cyclic FFE methodaccording to an embodiment of the present invention.

FIG. 7 illustrates anion and cation migration for the zoneelectrophoresis mode of an embodiment of the present invention.

FIG. 8 illustrates migration of particles of various isoelectric points(PI) for the isoelectric focusing mode of an embodiment of the presentinvention.

FIG. 9 describes various profiles of an embodiment of the presentinvention, wherein FIGS. 9A, 9B, and 9C depict voltage, media flowvelocity, and media displacement, respectively.

FIG. 10 describes various profiles of another embodiment of the presentinvention, wherein FIGS. 10A, 10B, and 10C depict voltage, media flowvelocity, and media displacement, respectively.

FIG. 11 describes various profiles of yet another embodiment of thepresent invention, wherein FIGS. 11A, 11B, and 11C depict voltage, mediaflow velocity, and media displacement, respectively.

DETAILED DESCRIPTION

The present invention and its advantages are further illustrated in thefollowing, non-limiting examples. In the drawings, like referencecharacters generally refer to the same parts throughout the differentviews. Also, the drawings are not necessarily to scale, emphasis insteadbeing placed upon illustrating the principles of the invention.

The general method of the present invention is reflected in FIG. 2(specifically FIGS. 2 a-2 e) while the elements typically required forcarrying out the invention are collectively described in FIGS. 3, 4, 7a, and 8 a. Reflected schematically in FIGS. 3 and 4 are two embodimentsof an electrophoresis apparatus 10 including an electrophoresis chamber12 as well as elements relating to and/or connected to theelectrophoresis chamber 12, while FIGS. 7 a and 8 a reflect specificallythe electrophoresis chamber 12 as well as the electrophoretic behaviorof the particles as schematically represented in the electrophoresischamber 12.

An apparatus 10 that may carry out aspects of the invention comprisesall or a portion of the following elements:

an electrophoresis chamber 12,separation media inlets 36,sample inlet(s) 38,electrodes (cathode 24 and anode 26),a power supply (not shown),a first fluidic displacement system 60,a second fluidic displacement system 61,controller(s) for controlling the power supply and/or the fluidicdisplacement systems.

Additionally, the following elements are typically desired to optimizethe control, flexibility, and performance of embodiments of theinvention:

cathode media 170,anode media 172,electrode/media spacer or barrier,chamber spacer 22,temperature controller 174,cooling element 178,sample outlets 52.

The above components and how they interrelate as a system will bedescribed specifically herein below, but first the general methodaccording to embodiments of the present invention is illustrated withreference to FIG. 2.

In the initial state shown on FIG. 2 a, stage 0 begins with the sampleand separation media introduced into the separation space of theelectrophoresis chamber. No voltage or, optionally, an ineffectivevoltage is applied between the electrodes. In stage 0, there is anineffective environment for successful electrophoretic migration tooccur. This could be as a result of zero or an otherwise ineffectiveamount of electrophoretic field strength between the electrodes, oroptionally due to rapidly introducing the sample and separation mediuminto the chamber thereby not providing enough residence time foreffective electrophoretic migration to occur.

Stage 1 shown in FIG. 2 b begins by reversing the flow rate of thecombined sample and separation media in cycles of forward and reversemovement in no particular order. Preferably, during a portion of stage1, an electrophoretic field is not causing electrophoretic migration tooccur. The displacement of the sample particles of interest is generallyless than the length of the electrodes. The voltage is increased orapplied between the electrodes to thereby produce an effective fieldstrength for electrophoretic migration to take place. Typical fieldstrengths applied are in the range of about 250 V/cm, although thespecific field strength used in a separation experiment will bedependent on a number of factors. Selecting an optimal voltage forcyclic FFE operation in a given FFE apparatus is well within the skillsof the person skilled in the art.

The next stages 2 and 3 are shown in FIG. 2 c. An effectiveelectrophoretic field is causing the particles to electrophoreticallymigrate due to the voltage applied between the electrodes. It should benoted that in stages 2 and 3, the sample particles of interest arealways in between and subjected to the electrophoretic field between theelectrodes since the displacement of the sample and separation mediasufficiently maintains the sample particles of interest to stay withinthe length of the electrodes.

It should be noted that during stages 2 and 3, the ability for thesample to electrophoretically migrate depends on the type ofelectrophoretic process employed by the user (i.e., isotachophoresis,isoelectric focusing, or zone electrophoresis), as well as theelectrophoretic mobility of the sample in the separation media which isheavily influenced by the characteristics of the separation media chosenas well as the ability for current to flow between the electrodes. Itshould also be noted that stage 2 and stage 3 differ only in directionof flow of sample and separation media during electrophoretic migration,and can be repeated multiple times with similar or different flowcharacteristics. An example would be to move the sample and separationmedia during stage 2 with a certain average fluidic velocity and duringstage 3, to move them backwards with an average fluidic velocity that isa fraction of or a multiple of the average fluidic velocity in stage 2.

When stages 2 and 3 are no longer performed, stage 4 may take place asshown in FIG. 2 d. Essentially, stage 4 involves displacing the sampleto be eluted towards sample outlets. While in FIGS. 2, 7 a, and 8 a thesample outlets are disposed at the opposite side of the electrophoresischamber, in other embodiments, the sample outlets may be on the sameside as the sample inlets and separation media inlets.

In FIG. 2 d, stage 4 involves essentially minimal, preferably noelectrophoretic migration of the sample during elution to the sampleoutlets. This can be accomplished by reducing or eliminating theelectric field between the electrodes by lowering or removing thevoltage between the electrodes. Optionally, this can be accomplished byquickly forcing the sample and separation media towards the sampleoutlets at an average fluid velocity that minimizes the duration andtherefore the impact of an electrophoretic field on the migration of theparticles or sample prior to reaching the sample outlets. It should berecognized that stage 4 is easiest performed by having no voltageapplied during elution or extraction of the electrophoreticallyseparated sample from the electrophoresis chamber 12. It will beappreciated that the above is particularly useful for zoneelectrophoresis FFE or isotachophoresis FFE applications. However,removing or reducing the voltage is not important when performing FFE inisoelectric focusing mode (IEF) since the total charge or net surfacecharge of the particles is zero at their final position between theelectrodes with the consequence that no further migration is observedupon a continued application of an electric field.

Stage 4 as shown in FIG. 2 d demonstrates elution of the sample andseparation media, thereby enabling further analysis or processing of theeluted sample. Stage 5, shown in FIG. 2 e which may occur in additionalembodiments simultaneously with stage 4 (FIG. 2 d) or at a time afterstage 4 terminates, thereby enabling further electrophoretic separationto occur for additional samples or electrophoretic environments. WhileFIG. 2 demonstrates basic principles of embodiments of the presentinvention, other aspects of the invention will be shown throughout theapplication.

Finally, stage 5 is carried out as shown in FIG. 2 e, essentially arepeat of stage 0 shown in FIG. 2 a. Optionally, FIG. 2 e may be avoidedor abandoned if only one electrophoresis process is needed by the user.Alternatively, stage 5 can be combined with stage 4 as long as mixing ofthe new sample with the separated sample from the previous run isprevented.

As shown in FIGS. 9, 10 and 11, different voltage, particle flowvelocity, and particle displacement profiles may exist that correspondto a sample being subjected to steps or stages 0 through 4 of theembodiment as reflected in FIG. 2. For the purposes of FIGS. 9 to 11,the Y direction may correspond to the direction from the inlet end walltowards the outlet end wall. FIGS. 9 to 11 are similar in many regards,with exception to the acceleration and deceleration of the media flowvelocity. In FIG. 9 b, it is apparent that the method of the inventionmay be carried out by applying the voltage needed for electrophoreticmigration to occur while the sample is moving forward (in this case,along the Y direction), while in FIG. 10 b, the sample is brought to astop prior to applying the voltage needed for electrophoretic migration.In FIG. 11, the reversal of the sample and separation media flow isinstantaneous (e.g. when using peristaltic pumps where the direction ofthe pump is switched very fast, see FIG. 11 b), although a slower andgentler reversal of the flow is certainly desirable to avoid anyturbulences affecting the separation quality.

It should be noted that while FIGS. 9 to 11 are representative ofcertain embodiments of the invention, many other conditions of voltage,media and sample flow rate, and sample displacement are possible and canbe appreciated by one skilled in the art. Additionally, the number ofcycles able to be achieved for other embodiments of the invention may bealtered, either decreased or increased, and can therefore provide theuser with flexibility for carrying out free-flow electrophoresis in acyclic interval mode of operation.

While FIG. 2 outlines the general mode of an embodiment of the inventionand FIG. 2 c outlines the general electrophoretic separation step ofembodiments of the invention, it should be noted that each of the threeprimary electrophoresis processes may be employed (i.e.,isotachophoresis, zone electrophoresis, and isoelectric focusing) as theelectrophoretic separation step. This is shown in more detailschematically in FIGS. 7 a and 8 a for zone electrophoresis andisoelectric focusing, respectively.

During continuous operation, band widening comes about due to thelaminar flow cross section, which results in higher retention times forthe analyte in the electrical field, and hence a stronger lateralmigration in the area of the electrophoresis chamber walls. Thistriggers an additional sickle-shaped band widening that is superposedover band widening via diffusion.

In contrast to the above, during cyclic interval operation where theelectrical field acts on an analyte, band widening is caused solelythrough diffusion, and hence is lower than in cases of continuousoperation. Lower band widening leads to a higher resolution. In otherwords, resolution is better in cyclic interval operation than incontinuous operation (CFFE). Thus, the cyclic interval free-flowelectrophoresis yields enhanced resolution of separation compared tocontinuous free-flow electrophoresis. Moreover, cyclic intervaloperation may achieve the same or better level of resolution whencompared to static interval free-flow electrophoresis, yet in a muchshorter time frame. This is due to the higher field strength that can beapplied if the sample and separation media is in motion compared to theprior art interval FFE where the electromigration occurred statically(flow turned off).

In other embodiments of the invention, the method can be combined withother variations of free flow electrophoresis processes and devices. Forexample, multiple devices or setups can be used, which are then arrangedin parallel and/or in series, for example as described in co-pendingapplication US 2004/045826 to Weber. In such combinations, the otherelectrophoresis processes may be also carried out in the cyclicinterval, static interval mode or continuous mode as described herein.By selecting the appropriate operation modes (cyclic interval, intervalor continuous) and separation modes (ZE, IEF and/or ITP), powerfulseparations of a variety of different particles may be achieved.

As shown in FIGS. 3, 4, 7 a and 8 a, an electrophoresis apparatus 10that may carry out aspects of the invention comprises an electrophoresischamber 12 defined by a floor or bottom plate 16, a cover or top plate14, end walls 18, 20 and side walls 56, 58. The end walls 18, 20 andside walls 56, 58 preferably form a substantially rectangularelectrophoresis chamber 12 structure interposed and supporting theopposed parallel top and bottom plate (14 and 16).

A plurality of electrodes located in electrode spaces, i.e., cathodespace 28 and anode space 30, are arranged proximate and parallel to thefirst and second side walls (56 and 58) within the electrophoresischamber 12 so that the electrodes are generally parallel to one another.While it is not absolutely necessary that the electrodes are exactlyparallel to each other, exact parallelism of the elongated electrodes ispreferred in order to provide a homogenous electric field throughout theseparation space within the electrophoresis chamber 12. Generally, ananode 26 is disposed in the anode space 30 and a cathode 24 is disposedin the cathode space 28. The electrodes (24 and 26) are typicallyseparated from the electrophoresis chamber 12 by electrode spacers 32which maintain a barrier to prevent electrophoretically separatedparticles from reaching, fouling, or otherwise interfering with theelectrodes during operation. Typically, the electrode spacers 32 areconstructed in the form of membrane electrode spacers 48 that areessentially filter membranes preventing exchange of media caused byhydro-dynamic flow. The membranes are typically located very close tothe electrodes, but for clarity the drawings show the membranes spacedfrom the electrodes. A more detailed depiction of the above can be foundin FIGS. 7 a and 8 a. The cross-sectional views shown in FIGS. 3 and 4depict a cross-section cut through the electrophoresis chamber 12 in adirection shared by the electrodes, generally normal to the top plate 14and bottom plate 16 as well as the inlet end wall 18 near the separationmedia inlets 36 which are described below.

A separation space or zone 34 is generally defined or delimited as thespace between the electrode spaces (28 and 30) and top and bottom plate(14 and 16) not including the electrode spacers 32. The separation space34 is flanked by the anode 26 and the cathode 24 which generate anelectric field when connected to a power supply (not shown). Preferably,the direction of the electric field is substantially parallel to the topand bottom plate (14 and 16), and is preferably also substantiallyperpendicular to the direction of the displacement of the bulkseparation medium within the electrophoresis chamber 12.

Preferably, the top and bottom plate (14 and 16) are stationary withrespect to the electrodes and each other, at least when voltage isapplied to the electrodes, so that the forces caused by the displacementflow and the electric field act on the sample and the ions in theseparation medium only.

Typically, the power supply is a direct current (DC) power supply withan AC to DC converter supplying current between the electrodes in agenerally controlled manner. Additionally, a controller may exist tocontrol the flow of current between the cathode 24 and anode 26.

The electrodes (cathode 24 and anode 26) are typically composed of ametal such as platinum that will not easily be oxidized in the electricfield. The electrodes (24 and 26) are optionally washed constantly by asalt or buffer solution to remove electrolysis products that are createdduring the process. The solution or media herein is generally referredto as cathode media 170 and anode media 172.

The bottom plate 16 and the top plate 14 of the electrophoresis chamber12 can independently be made of glass (preferably polymer-coated glass),or suitable plastics, such as PVC, polycarbonate, Plexiglas,polyhalohydrocarbons, or Lucite® (an acrylic resin consistingessentially of polymerized methyl methacrylate).

The top and bottom plate (14 and 16) are typically separated by chamberspacers 22 (not shown) that act as gaskets or seals. The chamber spacers22 usually delimit the separation space 34 within the electrophoresischamber 12. The separation space 34 (space between plates) usually has athickness of about 0.01 to about 1.5 mm, preferably of about 0.05 toabout 1 mm and most preferably of about 0.1 to about 0.5 mm. It will beappreciated that the thickness of the separation space depends on manyfactors, including the size of the electrophoresis chamber 12 and thesample volume to be separated or detected and may be adapted accordinglyby those skilled in the art.

An apparatus 10 according to embodiments of the present inventioncomprises elements to help introduce and extract the sample andseparation media into and from the electrophoresis chamber 12. Integralto the electrophoresis chamber 12 at its lower end wall are, forexample, a plurality of separation media inlets 36, typically havingports (not shown) which are connected by conduits as separation mediainlet tubing 74, such as flexible TEFLON tubes. The tubes are in fluidiccommunication to feed channels of a fluidic displacement system 60(e.g., a multi-channel pump such as a peristaltic pump). The separationmedia inlets are generally arranged collinear at the lower end of theelectrophoresis chamber 12 and equidistant with respect to thecorresponding outlets, as illustrated, e.g. in FIGS. 7 a and 8 a. Sampleinlet tubing 72 conduits for introducing and eluting or expelling thesample and related media and buffers into and out from theelectrophoresis chamber 12 are shown in FIGS. 7 a and 8 a and areschematically shown in FIGS. 3 and 4. The separation media inlets 36 aresupplied with a separation medium while the electrode inlets (40 and 44)are fluidically connected to the electrode spaces (28 and 30) andsupplied with the electrode stabilization medium for the anode 26 andcathode 24. The sample containing the analytes is injected into apredetermined position within the separation space 34 through sampleinlet 38. Wherein multiple separations are capable of being performedsimultaneously and parallel to each other between one or more pairs ofelectrodes or conductivity walls, it is contemplated that multiplesample inlets 38 can be used simultaneously to introduce the correctsample or samples into the separation space 34. In this case, themultiple sample inlets are also positioned collinear and equidistant tothe outlet end as described above for embodiments comprising multipleseparation media inlets.

Alternatively, the separation media inlets 36 and sample inlets 38 canbe combined. In this embodiment, the sample may be introduced throughthe separation inlets, for example injecting the sample through an inletport in the portion of the tubing 74 external to the electrophoresischamber 12.

An apparatus 10 according to embodiments of the present invention maycomprise as many as three fluidic displacement systems. A first fluidicdisplacement system controls the displacement of the sample andseparation media in the separation space 34 as will be described indetail below. Typically, the first fluidic displacement system is amulti-channel peristaltic pump but may take on the form of other pumpssufficient to displace fluid in a controlled manner within theseparation space 34.

A second fluidic displacement system serves two purposes, namely theintroduction of the sample into the separation space 34 and theintroduction and displacement of the separation media into and withinthe separation space 34. The second fluidic displacement systemcomprises two multi-channel peristaltic pumps, one for the introductionof the sample into the separation space 34 and one for the introductionand displacement of the separation media and the sample. It however ispossible to have one multi-channel peristaltic pump perform both tasks.The use of multi-channel peristaltic pumps is preferred, but the secondfluidic displacement system may take on the form of other pumpssufficient to displace fluid in a controlled manner within theseparation space 34.

The second fluidic displacement system may also perform the task ofintroducing and displacing a counterflow media into and within theseparation space 34 as will be described below in detail. It however isalso contemplated to use a further multi-channel pump for this purpose.

Finally, a third fluidic displacement system (not shown) may be utilizedfor circulating the electrode media within the electrode spaces.

The fluid displacement systems interface with the various types of mediain a controlled fashion. A controller (not shown in the figures) istypically utilized to manage the flow rates and pressures of the abovefluids to carry out the desired separation or fractionation protocolchosen by the user.

In an embodiment of an apparatus capable of carrying out the inventionshown in FIG. 3, two fluid displacement systems 60 and 61 or pumps areutilized. A first fluidic displacement system 60 is utilized to providebulk fluid displacement of the sample and separation media between theelectrodes along the direction of the electrodes. A second fluidicdisplacement system 61 is used to introduce the sample and separationmedia into the separation space 34, either together or separately, andto initially displace both within the separation space 34. In apreferred embodiment, the second fluidic displacement system 61comprises two fluidic displacement units, one for the introduction ofthe sample into the separation space and one for the introduction anddisplacement of the separation media. This second fluidic displacementsystem 61 is also used to remove or elute the sample afterelectrophoretic separation from the separation space 34 through sampleoutlets 52. Separate valving may be utilized to enable or prevent theintroduction of the sample through sample inlet 38 into the separationspace as desired by the intended operation of embodiments of theinvention.

An inlet end cycle conduit 86 and an outlet end cycle conduit 88 areoperatively connected to the first fluidic displacement system 60 andare additionally fluidically coupled to the separation space 34 of theelectrophoresis chamber 12. Their use is related to cycling of theseparation media and samples back and forth along the direction of theelectrodes rather than introduction of the separation and sample mediainto the separation space 34.

The first fluidic displacement system 60 provides a displacement forceor pressure for controlling the bulk fluid flow of sample and separationmedia after they have been introduced into separation space 34. Forexample, once separation space 34 and the inlet and outlet cycle tubesare filled with media or a buffer solution, operation of the firstfluidic displacement system 60 (in this case, a peristaltic pump),transfers the sample and separation media towards one end of theelectrophoresis chamber 12, and reversing the operation of the firstfluidic displacement system 60 causes the sample and separation media tomove towards the other end of the electrophoresis chamber 12. Desirably,the operation of the first fluidic displacement system 60 will bereversed prior to the sample reaching either end of the separation space34. More desirably, the operation of the first fluidic displacementsystem 60 will ensure that the sample does not leave that portion of theseparation space 34 that is between the electrodes.

The second fluidic displacement system 61 (e.g. in the form of twoperistaltic pumps) is connected via sample inlet tubing 72 to the sampleinlet 38. Also connected to the second fluidic displacement system 61 isseparation media inlet tube 74 that is fluidically coupled to separationmedia inlet 36. The second fluidic displacement system 61 controls thedelivery of sample and separation media into the separation space 34 aswell as controls the extraction of fractionated sample from theelectrophoresis chamber 12 into individual collection wells, vessels orcavities 82 in for instance a microtiter plate 80.

It should be noted that desirably, when second fluid displacement system61 is in operation, first fluidic displacement system 60 is not and viceversa.

In operation, the embodiment reflected in FIG. 3 is operated as follows.The second fluidic displacement system 61 causes the introduction of thesample and the introduction of the separation media into the separationspace 34 as well as the advancement of both the sample and theseparation media into the center region of the displacement space 34.Preferably, electrophoretic migration does not occur during this stage.Next, the first fluidic displacement system 60 moves the sample andseparation media back and forth along the direction of the electrodes bydisplacing fluid through the inlet end cycle conduit 86 and the outletend cycle conduit 88. During a portion of this movement or cycling, anelectrical current flows between the electrodes thereby causing orenabling electrophoretic migration. After sufficient cycling andelectrophoretic migration, the first fluidic displacement system 60preferable is no longer used or is turned off, and the second fluidicdisplacement system 61 causes additional separation media to beintroduced into the separation space thereby displacing the separatedsample from the separation space, through sample outlets and intopreferably collection vessels such as those found in a microtiter plate.It may be possible during this elution phase to also introduceadditional sample into the separation space.

Also, it may be possible to introduce counterflow media into the outletend wall 20 through counterflow inlets 54, wherein the sample outlets 52are generally interposed between the sample media inlets 38 and thecounterflow inlets 54. During the elution phase or step, it is desirablethat the high voltage is off or at least ineffective to causeelectrophoretic migration, particularly for the ITP and ZE operationmodes. For obvious reasons, turning off the voltage is not necessarywhen the separation is carried out in IEF mode (see below). It should benoted that valving of the inlets, outlets, or tubing conduits involvedin this embodiment may facilitate the process steps as described above.

In an additional embodiment of an apparatus capable of carrying out theinvention, one fluidic displacement system 62 is utilized forcontrolling the movement of fluids to carry out electrophoreticseparation in a cyclical interval manner. A first pump of the fluidicdisplacement system 62 is connected via sample inlet tubing 72 to thesample inlet 38. A second pump of the fluidic displacement system 62 isconnected to the separation media inlet tube 74 that is fluidicallycoupled to separation media inlet 36. The fluidic displacement system 62controls the delivery of sample and separation media into the separationspace 34 as well as the movement of fluids by providing a displacementforce or pressure for controlling the bulk fluid flow of sample andseparation media after they have been introduced into separation space34. For example, once separation space 34 and the inlet and outlet cycletubes are filled with media or a buffer solution, operation of thefluidic displacement system 62 (in this case, a peristaltic pump),transfers the sample and separation media towards one end of theelectrophoresis chamber 12, and reversing the operation of the fluidicdisplacement system 62 causes the sample and separation media to movetowards the other end of the electrophoresis chamber 12. Desirably, theoperation of the fluidic displacement system 62 will be reversed priorto the sample reaching either end of the separation space 34. Moredesirably, the operation of the fluidic displacement system 62 willensure that the sample does not leave that portion of the separationspace 34 that is between the electrodes.

After electrophoretic separation has occurred, the net flow of sampleinto and out of the separation space 34 is held constant by maintainingthe volumetric flow of media introduced by the counterflow inlets 54 andthe separation media inlets 36 to each be zero or positive, wherein whenthe sum of both of the flows is positive, the fractionated sample iscapable of eluting through the sample outlets 52, through the outlettubing 76 and into preferably individual collection vessels or cavities82 disposed in, for instance, a microtiter plate 80.

Optionally the second pump of the fluidic displacement system 62 mayalso be connected to a counterflow tubing 78 fluidically coupled via acounterflow inlet 54 into the separation space 34.

In addition to the separation media inlet tubing 74, separate valvingmay be utilized to enable or prevent the introduction of the samplethrough sample inlet 38 into the separation space 34 as desired by theintended operation of embodiments of the invention.

The embodiment depicted in FIGS. 4 a and 4 b operates as follows. Thefluidic displacement system 62 causes the introduction of the sample andseparation media into the separation space 34. In a preferredembodiment, the fluidic displacement system 62 comprises two fluidicdisplacement systems, one for the introduction of the sample into theseparation space and one for the introduction and displacement of theseparation media. Preferably, electrophoretic migration does not occurduring this stage. Next, the fluidic displacement system 62 moves thesample and separation media back and forth along the direction of theelectrodes. This is performed such that operation of fluid displacementsystem 62 causes the separation media and the sample to shift forwardand backward, towards and away from the inlet end wall 18 in a cyclingmotion. In variations of the embodiment depicted in FIGS. 4 a and 4 b,both the counterflow tubing 78 and separation media inlet tubing 74 areengaged with fluidic displacement system 62 to cause pressuredifferentials between the counterflow inlets 54 and the separation mediainlets 36.

During a portion of the movement or cycling, an electrical currentflowing between the electrodes causes or enables electrophoreticmigration. After sufficient cycling, electrophoretic migration or both,the fluidic displacement system 62 is used to elute the separated samplefrom the separation space 34 through sample outlets 52 and intopreferably collection vessels or cavities 82 such as those found in amicrotiter plate 80. It may be possible during this elution phase toalso introduce additional sample into the separation space. Also, it maybe possible to introduce counterflow media near the outlet end wall 20,wherein the sample outlets 52 are generally interposed between thesample media inlets 38 and the counterflow inlets 54. During the elutionphase or step, it is desirable that the high voltage is off or at leastineffective to cause electrophoretic migration at this point. It shouldbe noted that valving of the inlets, outlets, or tubing conduitsinvolved in this and other embodiments may facilitate the process stepsas described above.

During the cycling of the sample flow, it is essential by any means toprevent the entry of sample or media into the sample outlet as well asthe entry of air into any of the inlet tubing. For example, in theembodiment depicted in FIGS. 4 a and 4 b, it is desirable to have thesample outlets resist or prevent the entry of sample or media into theoutlets during cycling of the sample flow by closing a valve, causing apressure head to develop in the outlets by elevating the outlet tubing76 with respect to the separation space 34, or constricting the outlettubing such that there is more fluidic resistance for the separationmedia to enter the outlet tubing 76 than that of the counterflow tubing78, sample inlet tubing 72, or separation media tubing 74. Other meansof accomplishing the same are to be understood by one skilled in theart.

An apparatus 10 according to embodiments of the present invention mayfurther contain a fraction collector outlet or sample outlet 52 andoutlet tubing 76. In most cases of operation, typically afterelectrophoretic separation according to at least one of the abovementioned electrophoresis modes, the separated fractions, i.e., analytesof interest, are collected through the spatially distinct sample outlets52 arranged in a line and in proximity to an outlet end wall 20,typically opposite an inlet end wall 18 adjacent to the separation mediainlets 36. The analytes or fractions are led through outlet tubing 76 orconduits to individual collection vessels of any suitable type,preferably cavities 82 or wells in microtiter plates 80 (see FIGS. 3 and4). In the collection vessels, the analytes are collected together withthe separation medium and, optionally, the counter-flow medium. Thedistance between the individual sample outlets 52 of the array ofcollection outlets should generally be as small as possible in order toprovide for a suitable fractionation/separation. The distance betweenindividual sample outlets 52, measured from the centers of thecollection outlets, is typically from about 0.1 mm to about 2 mm, moretypically from about 0.3 mm to about 1.5 mm, and is almost alwaysdependent on the maximum diameter of the outlet tubing 76. This seriesof tubing openings with the smallest possible distance between theopenings is referred to as a fractionating device, and the number ofthese outlet tubes arranged in parallel ranges is between 2 and amaximum of 1000, preferably between 30 and 200, and more preferablybetween 20 and 100. The number, shape, and location of these outlets maybe modified in different modes to purposefully fractionate or isolate aparticular portion of the separated sample according to a separationgradient.

The selected sample components or fractions collected may be used foranalytic or preparative purposes. Alternatively, the fractions may bedirectly or indirectly introduced into the same or optionally a remoteelectrophoretic device to carry out additional processing. Such mayoccur in the event of performing a coarse fractionation followed by afine (higher resolution) fractionation, or in one or multipleelectrophoretic modes (ZE, IEF, ITP, or combinations thereof).

Extraneous sample components that are not collected through thecollection outlets through which the desired sample is withdrawn orwhich are collected through different collection outlets can also bediscarded.

In various embodiments of the invention, the separated sample may beintentionally diluted during the fractionation step or alternatively,may be extracted from the chamber by use of a counterflow process. Inthe counterflow process, a counterflow medium 176 may be introducedthrough counterflow inlets 54 generally opposing the direction of bulkflow of sample during elution, for the sample and separation media inthe separation space 34 to be selectively extracted from the separationspace 34. The sample and separation media unite with the counterflowmedia at the extraction or sample outlets 52, and are extractedtherethrough. The counterflow serves to enhance resolution of separationby allowing adjustment and control of the flow and pressure conditionsat the collection outlets, thereby maintaining control for the desiredfraction to reach and be extracted through the desired sample outlet 52.Details of the counterflow process are disclosed, e.g., in U.S. Pat. No.5,275,706 to Weber. In view of the above-mentioned advantages, using acounterflow is generally preferred, although in certain situations, suchcounterflow may not be desired, e.g., due to the inherent dilutioneffect.

In addition to the use of counterflow depicted in FIGS. 4 a and 4 bdescribed above, it is possible to use a counterflow technique in theelectrophoresis chamber 12 according to an embodiment of the inventionduring sample elution. For example, in FIGS. 7 a and 8 a, a counterflowmedium may be introduced during an elution step where the sample hasbeen subjected to electrophoretic migration in the electrophoresischamber 12 via counterflow tubing 78 towards and through counterflowinlets 54. The counterflow medium is introduced in a direction oppositethe direction in which the sample and separation medium is eluted fromthe electrophoresis chamber 12. The counterflow enhances separation byallowing adjustment and control of the flow and pressure conditions atthe sample outlets 52 and reduces the occurrence of any voids that mayimpact sample resolution during elution. For details of how to usecounterflow, reference is again made to U.S. Pat. No. 5,275,706 which isincorporated herein by reference in its entirety.

The counterflow media, if used, are typically selected to be capable ofmodifying or surpassing the buffering capacity of the separation mediumapproaching the sample outlets 52, and may therefore be a materialhaving the same physical properties such as viscosity and density butdiffering in conductivity and/or pH-value and/or their chemicalingredients. Typical counterflow and separation media are selected fromthe same group of media, and may contain components such as urea,glycerol, carbohydrates, and similar compounds.

A useful ratio of the flow rate of the separation medium to the flowrate of the counterflow medium is about 1:10 to about 10:1, moretypically about 1:3 to about 3:1. The actual flow rates for theseparation medium and the counterflow media depend on a variety ofconsiderations, including the geometrical dimensions of the apparatus10, the particular separation mode used (which may vary in requiredtransit time), the sample to be separated, the separation medium usedand the counterflow medium or media used in order to obtain an optimumseparation of the analytes. Typical flow rates of all media into thesystem (stabilization and counterflow) may therefore range widely from0.3 mL/hour to 3000 mL/hour, depending on the circumstances and thegeometry of the electrophoretic chamber. The linear velocity of thesample and separation medium is, however, generally between 0.01 and 50mm/sec, and preferably between 0.1 and 10 mm/sec.

Temperature control is also useful in the embodiments of the presentinvention. When current passes through an electrolyte solution, thetemperature of the conduction medium increases, according to thephenomenon known as Joule heating which may influence viscosity and/orfluid flow through the chamber, thereby causing flow disturbances. Toreduce disturbances affecting the laminar flow profile of the flowingmedium caused by such heating, it is generally desirable to dissipatethe Joule heat to the surroundings. Many ways to practically achieve thedesired cooling will be apparent to the person skilled in the art. Forexample, a cooling element 178 (not shown) may be utilized to carry outsuch an effect. The electrophoresis chamber 12 may be arranged with itsbottom plate 16 on a metal support that contains fluid flow channelsconnected to the cooling element 178. The cooling element 178 may be atemperature control device 174, such as a Peltier or thermoelectric (TE)module. A thermostat closed loop control system for measuring andcontrolling the temperature of the electrophoresis chamber 12 is usefulto maintain the desired temperatures for the separation space 34. Usefultemperature ranges are from about 2° C. to about 35° C., more typicallyfrom about 5° C. to room temperature (about 20-25° C.).

In embodiments of the invention wherein a membrane electrode spacer 48isolates the cathode and anode spaces (28 and 30) from the separationspace 34, the electrode spaces (28 and 30) may be purged by circulatinga flow of cathode media 170 and anode media 172 (together referred to aselectrode media) at a high flow rate using a pump. The electrode mediamay be cooled and reintroduced into the electrode spaces (28 or 30) in acontinuous manner. How the electrode media is cooled is reflected inFIGS. 7 a and 8 a, wherein the cathode media enters into the cathodemedia inlet 44 and exits via the cathode media outlet 46. Before thecathode media or anode media re-enter the electrophoresis chamber 12,they may be subjected to a cooling element 178 such as a heat-transferelement that removes thermal energy from the anode and cathode mediaprior to reintroduction.

Specifically, as reflected in FIGS. 7 a and 8 a, the anode media 172enters into the anode media inlet 40 and exits via the anode mediaoutlet 42. In embodiments wherein the electrode spacer 32 is a physicalbarrier such as membrane electrode spacer 48, the flow rate of thecathode and anode media (170 and 172) can vary from the flow rate of thebulk sample and separation media.

It is readily apparent that other suitable ways for cooling theelectrode media exist and can be applied by the person skilled in theart.

The controllers mentioned above for the power supply and fluidic controlmay operate as one unit, or optionally as independent units. Each may becontrolled by the user independently of one another. Setting up theparameters of the protocol(s) processed in the controller(s), such asmode of electrophoresis, number of cycles, retention time of a cycle,etc., is preferably done by means of a software program providing agraphical user interface for entering the parameter values.

Accordingly, in another aspect, the present invention relates to acomputer executable software code stored on a computer readable medium,wherein the code is suitable for effectuating the separation ofparticles in an electrophoresis chamber 12, wherein the electrophoresischamber has a top plate, a bottom plate, and a plurality of electrodesgenerally parallel to one another with a separation space disposedtherebetween, and one or more fluidic displacement systems for conveyinga separation medium between the electrodes, wherein the softwarecomprises code to enable selection and application of a voltage betweenthe electrodes effective to manipulate particles by electrophoresis,code to enable displacement of at least a portion of the separationmedium and sample toward a first direction parallel to the direction ofthe electrodes, and code to enable displacement of at least a portion ofthe separation medium and sample toward a second direction opposite thefirst direction; and code to enable displacement of the at least aportion of the separation medium and sample toward the first direction.In other words, the software program supports a user in selectingadequate parameter values for the intended separation process, includingvoltage, current, flow rates of separation medium, sample, andoptionally counterflow medium as well as the number of cycles. This istypically accomplished by providing the user with forms to be filled in,which adapt their layout, e.g., the information and input fields shown,to the parameter values entered so far, i.e., selected mode ofelectrophoresis, etc. Additionally, the software program may verify theentered parameter values and inform the user about parameter valuesbeing incorrect or out of range. Furthermore, the software program maysupport the user by automatically providing parameter values, which canbe derived from already established parameter values, e.g., totalretention time after having entered the number of cycles and theretention time for each cycle.

Desirably, all media flow through the separation space 34 is done sounder laminar flow conditions. As shown in FIGS. 3, 4, 7 a, and 8 a, thedirection of flow from the various inlets (36 and 38) while filling theelectrophoresis chamber 12 with the sample or with the separation mediais shown by arrows for various embodiments of this invention.

Different modes of electrophoresis may be employed to influence particlemigration based upon, for example, the particle's isoelectric point(i.e., isoelectric focusing of zwitterions (IEF)), or optionally basedon the particles net charge (i.e., zone electrophoresis (ZE) forparticles with different or varying net charges). Particles can also beseparated based upon their electrophoretic mobilities when interposedbetween leader (fast moving) and trailing (slow moving) buffer systems(i.e., isotachophoresis (ITP) for anionic or cationic particles).

In a first mode of electrophoretic separation, a zone electrophoresis(ZE) FFE separation technique is carried out. Zone electrophoresis isbased on the difference between the electrophoretic mobility value ofthe particles to be separated and that of the separation mediumemployed. FFE zone electrophoresis makes it possible to isolate analytesand particles on the basis of differing size and/or form and/or netsurface charge.

The ZE FFE method is particularly suitable for separating “sensitive”bioparticles and complexes when specific demands have to be placed uponthe separation medium during separation. This is typically the case whenthe biological function and integrity of the particles has to bemaintained following separation. The special requirements in these casesare, e.g., a very restricted pH range for the separation medium, goodseparation medium buffer capacity, physiological compatibility of thebuffer substances used, and a minimum content of various “essential”cations and anions, etc.

FIG. 7 b shows a zone electrophoresis apparatus 10 with a flat pHprofile 134. A separation medium flows in a laminar manner between boththe electrodes as demonstrated within stage 2 and 3 of FIG. 2 c. Asample with three groups of particles (90 a, 104 a, and 118 a) to beseparated is introduced into the separation medium via the sample inlet38 and transported by the laminar flow of the separation medium. Thethree groups of particles are separated during stage 2 and 3 of the stepas reflected in FIG. 2 c, and are thereby allowed to migrate and becollected in distinct fractions in the pH gradient resulting from theelectrical field generated between the electrodes in the separationmedium as is indicated in FIG. 2 d. Specifically, the paths of firstparticle 90 a, second particle 104 a, and third particle 118 a arereflected in FIG. 7 a. Each particle (90 a, 104 a, 118 a) follows aspecial path or movement within stages 2 and 3 of electrophoreticmigration. The particles have a net charge that influences theirmovement within the separation space under conditions that enableelectrophoretic migration within the separation media. Third particle118 a has a slightly positive net charge and therefore under zoneelectrophoresis conditions, slightly deflects towards the cathode whilethe bulk fluid flow of the separation media influences net particlemovement of third particle 118 a with respect to the inlet and outletends of the chamber. Second particle 104 a on the other hand has arelatively strong positive charge, and therefore during the path ofmovement under zone electrophoresis conditions, greatly deflects towardsthe cathode while the bulk fluid flow of the separation media influencesnet particle movement of second particle 104 a with respect to the inletand outlet ends of the chamber. First particle 90 a on the other handhas a relatively strong negative charge, and therefore during the pathof movement under zone electrophoresis conditions, greatly deflectstowards the anode while the bulk fluid flow of the separation mediainfluences net particle movement of first particle 90 a with respect tothe inlet and outlet ends of the chamber. It should be noted that FIG. 7a reflects a zone electrophoresis (ZE) separation wherein the particleswill continue to migrate towards the electrode that has an oppositecharge than the net charge of each respective particle in the presenceof an electrophoretic field. If more cycles of forward and backwardmovement occurred, or if a longer period or duration of electrophoreticmigration was allowed to take place, the particles would continue tomigrate until they reached the electrode or some other electrode spacer32 such as a membrane electrode spacer 48 or optionally a non-membraneelectrode spacer 50 such as a high conductivity wall or buffer. In allcases of the first, second, and third particles (90 a, 104 a, 118 a) thefourth path reflected in FIG. 7 a is represented by the elution stepwherein electrophoretic migration is not very influential or evennon-existent as a component of each particle's net direction.

FIG. 7 a shows such a ZE apparatus in which the samples are separated onthe basis of their charge and to a lesser extent on the basis of theirform and size. As indicated in FIG. 7 b, the electrical conductivityprofile 136 of the separation medium between the two electrodes includeda high conductivity region 138 in the cathode space 28 and anode space30, both of which are in the vicinity of the electrodes. These are shownin FIG. 7 b as high conductivity regions 138, while the separation mediais represented as a low conductivity region 140. The pH value is thesame throughout the entire separation medium. The flat pH profile 134 isgenerated in the separation medium between the cathode and anode mediabuffers 170 and 172, respectively. While high pH regions 144 existwithin the cathode and anode media buffers, a low pH region existstherebetween.

In a second mode of electrophoretic separation, separation is based onthe different pI value of the particles to be separated, with this pIvalue being equivalent to the pH value of the surrounding,non-homogenous medium against which the particles appear neutral. FFE ofparticles on the basis of different isoelectric points allows analytesor particles with only minimal disparities in their pI values to beisolated. This is termed isoelectric focusing (IEF) carried out infree-flow electrophoresis (FFE). At the isoelectric point pI (i.e. atthe point in the separation medium displaying the pH value at which thenumber of negative and positive charges is equal for a given particle,e.g. a protein molecule) the total charge or net surface charge of thisparticle is zero. The focusing effect inherent in the separation mediumof an apparatus causes a particle which is diffused away from pI toautomatically receive a (positive or negative) net surface charge and tobe transported in the direction of pI again by the electrical field.

As a result of the charge transfer (i.e., electrical interaction)between analytes or particles with ionic molecules (preferablymonovalent ions) of the separation medium, it is to be expected thatthere is a major change in the pI value and therefore a rapid and totalseparation of the complex consisting of the particle and the ionicmolecule from the rest of the sample species. The cyclic isoelectricfocusing technique is especially suitable for the preparative isolationof biopolymers in general as well as of bioparticles, the biologicalfunction or integrity of which is certain to be within the range of theselected span of pH gradients in the separation medium. By adding“osmotic expanders” it is possible to ensure that ideal osmotic pressureis maintained. For example, by adding uncharged substances (e.g. sucroseor mannitol, etc., as a non-ionic osmotic expander) and/or salts (e.g.NaCl as an ionic osmotic expander), optimal osmotic pressure (i.e.isomolal conditions) can be achieved for a given cell type (e.g.,250-310 mosmol for mammalian cells).

FIG. 8 b shows an IEF electrophoresis apparatus 10 with a linear pHprofile gradient 142. A separation medium flows in a laminar mannerbetween both the electrodes as demonstrated within stage 2 and 3 of FIG.2 c. A sample with three groups of particles (90 b, 104 b, and 118 b) tobe separated is introduced into the separation medium via the sampleinlet 38 and transported by the laminar flow of the separation medium.The three groups of particles are separated during stage 2 and 3 of thestep in FIG. 2 c, and are thereby allowed to focus and be collected indistinct fractions in the pH gradient resulting from the electricalfield generated between the electrodes in the separation mediumrepresented as stage 4 or FIG. 2 d. Specifically, the paths of firstparticle 90 b, second particle 104 b, and third particle 118 b arereflected in FIG. 8 a. Each particle (90 b, 104 b, 118 b) follows aspecial path or movement within stages 2 and 3 of electrophoreticmigration. The particles have a pI that influences their movement withinthe separation space under conditions that enable electrophoreticmigration within the IEF separation media. Third particle 118 b has a pIslightly higher than the pI of the media near its original position, andtherefore during the path of movement under IEF electrophoreticconditions, slightly deflects towards the cathode while the bulk fluidflow of the separation media influences net particle movement of thirdparticle 118 b with respect to the inlet and outlet ends of the chamber.Second particle 104 b on the other hand has a pI much higher than the pIof the media near its original position, and therefore during the pathof movement under IEF electrophoretic conditions, greatly deflectstowards the cathode while the bulk fluid flow of the separation mediainfluences net particle movement of second particle 104 b with respectto the inlet and outlet ends of the chamber. First particle 90 b on theother hand has a pI much lower than the pI of the media near itsoriginal position, and therefore during the path of movement under IEFelectrophoretic conditions, greatly deflects towards the anode while thebulk fluid flow of the separation media influences net particle movementof first particle 90 b with respect to the inlet and outlet ends of thechamber. It should be noted that FIG. 8 a reflects an IEF separationwherein the particles never reached their pls. If more cycles of forwardand backward movement occurred, or if a longer period or duration ofelectrophoretic migration was allowed to take place, the particles wouldeventually reach their pIs and only experience movement forward andbackward as would the bulk fluid flow of the sample and separationmedia. In all cases of the first, second, and third particles (90 b, 104b, 118 b) the fourth path reflected in FIG. 8 a is represented by theelution step wherein electrophoretic migration is not very influentialor even non-existent as a component of each particle's net direction.

FIG. 8 b indicates the electrical conductivity profile 136 of theseparation medium between the two electrodes with high electricalconductivity in the cathode space 28 and anode space 30 in the vicinityof the electrodes. These are shown in FIG. 8 b as high conductivityregions 138, while the separation media is represented as a lowconductivity region 140. The pH profile gradient 142 is generated in theseparation medium between the cathode and anode media buffers 170 and172, respectively.

Additionally, an apparatus 10 according to embodiments of the presentinvention can be carried out in the isotachophoresis mode (ITP). The ITPFFE separation technique is based upon the difference in theelectrophoretic mobility value of the particles to be separated. Incontrast to ZE FFE, separation is achieved in non-homogenous separationmedia and offers better dissolution due to the inherent “focusingeffect”. When single particles are diffused from a separated band ofparticles (e.g. proteins) during ITP these particles enter a medium withvarying electrical field strength, resulting in the particles beingaccelerated or decelerated. The inherent focusing effect means that theslower or faster moving particles find their way into the dominantfraction again. While not shown schematically as above for zone and IEFelectrophoresis techniques, the present invention shows tremendous valueand results when used in an ITP mode of operation.

It will be appreciated by those skilled in the art that variouscombinations of these separation techniques (IEF and ZE FFE or IEF andITP) are possible. For instance, it is possible to use differentseparation media at the same time within the electrophoresis chamber ofthe FFE apparatus, thereby employing different separation parameterssimultaneously.

In other embodiments of the invention, it is contemplated that thecyclic interval mode can be carried out in multiple separation subspaceswithin separation space 34 between a single pair of electrodes. Lowmobility boundaries (with high conductivity) between adjacent sub-spacesare defined by the adjacent flows of cathodic stabilization medium andanodic stabilization medium that are disposed between the subspaces. Allmedia are typically introduced through their respective inlets via apump such as a multi-channel peristaltic pump. For details of thismethod, it is referred to international application no. PCT/US06/016175,which is hereby incorporated by reference in its entirety.

Preferably, the separation medium is a matrix-free, aqueous medium. Thechoice of the buffer components depends on the application mode (ZE,IEF, or ITP) and the nature of the sample or the separation problem.Suitable buffer systems are generally known in the art, and are in someinstances commercially available (e.g., from BD GmbH, Planegg, Germany).Preferred buffer systems for carrying out embodiments of the presentinvention are, for example, described in co-pending applicationUS2004/101973, provisionally filed application U.S. Ser. No. 60/885,792,and U.S. Ser. No. 60/945,246, all of which are hereby incorporated byreference in their entirety. Particularly preferred for cyclic intervalFFE as described herein are simple buffer systems comprising one bufferacid and one buffer base, especially if the separation is carried out inZE or flat-gradient IEF mode.

In some embodiments of the invention, the anodic stabilization medium,which is typically aqueous, may for example comprise an acid selectedfrom the group consisting of gluconic acid, glucuronic acid,acetylsalicylic acid, 2-(N-morpholino) ethanesulphonic acid (MES), andzwitterionic buffers (also called Goods buffers—see Good et al.,Biochemistry 5, 467 (1966)). In some embodiments of the invention, thecathodic stabilization medium, which is typically aqueous, may comprisea base selected from the group consisting of N-methyl-D-glucosamine,tri-isopropanolamine and 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (BISTRIS).

One possibility of improving electrophoretic separation is to maximizethe period of time a sample is moving and interposed between an anodeand cathode. To accomplish this, the average fluid velocity, duration,and distance of displacement for the bulk flow of media and sample canbe adjusted to maximize the electrophoretic performance of theapplication. In some instances, for example, residence time might be ofa major concern and therefore the number of cycles as well as theduration, distance, and linear particle speed for the bulk flow ofseparation media may be adjusted to optimize output and performance. Inother applications, band broadening due to a laminar flow profile may beof interest to control. The flexibility of controlling the systemparameters allows the user to maximize output and fractionation qualitybased on the type of application he or she is utilizing. The cyclicinterval method according to the present invention provides theopportunity to perform any FFE-separation technique inside a singleelectrophoresis chamber 12. Moreover, the chamber may even be of shorterdimensions even at prolonged times of electrophoresis when compared tothe chamber dimensions required by continuous free-flow electrophoresismachines.

As mentioned above, the present invention is particularly suitable for,but not limited to, the analysis and preparative separation of ions,peptides, biopolymers, bioparticles such as vesicles, cell membranes,cell organelles, viruses, etc., as well as synthetic polymers andgenerally any particles that may be influenced by an electrophoreticfield.

It will be apparent to those of skill in the art that many modificationsand variations of the embodiments described herein are possible withoutdeparting from the spirit and scope of the present invention as definedby the appended claims.

1. A method for separating particles comprising: disposing a separationmedium and sample in an electrophoresis chamber having a top plate, abottom plate and a plurality of electrodes generally parallel to oneanother with a separation space disposed therebetween, and a fluidicdisplacement system for conveying a separation medium between theelectrodes; applying a voltage between the electrodes effective tomanipulate particles electrophoretically; displacing at least a portionof the separation medium and sample towards a first direction generallyparallel to the direction of the electrodes; displacing at least aportion of the separation medium and sample in a second directiongenerally opposite the first direction.
 2. The method of claim 1,further comprising the step of eluting the separation medium and samplefrom the electrophoresis chamber through one or more outlets.
 3. Themethod of claim 2, wherein at least a portion of the separation mediumand the sample is eluted from the separation space into individualcollection cavities such as vessels or wells in microtiter plates. 4.The method of claim 2, wherein at least a portion of the separationmedium and sample are eluted towards outlets by displacing theseparation medium and sample in a direction consistent with the first orsecond direction.
 5. The method of claim 2, wherein prior to eluting theseparation medium and sample from the electrophoresis chamber, themethod comprises removing the voltage between the electrodes effectiveto manipulate particles electrophoretically.
 6. The method of claim 1,wherein applying a voltage between the electrodes enables anelectrophoretic separation mode chosen from the group consisting ofisoelectric focusing, isotachophoresis, and zone electrophoresis.
 7. Themethod of claim 1, wherein the top plate and bottom plate are stationarywith respect to the electrodes and each other while applying thevoltage.
 8. The method of claim 1, wherein one cycle comprisesdisplacing the separation medium and sample towards a first directiongenerally parallel to the direction of the electrodes, and displacingthe separation medium and sample in a second direction generallyopposite the first direction; and wherein the number of cycles isgreater than one.
 9. The method of claim 8, wherein the number of cyclesis selected so as to achieve sufficient separation of the sample. 10.The method of claim 1, further comprising the step of introducing anddisplacing a counterflow medium into the separation space.
 11. Anapparatus for separating particles comprising: an electrophoresischamber having a top plate, a bottom plate and a plurality of electrodesgenerally parallel to one another with a separation space disposedtherebetween, a fluidic displacement system configured to introduce aseparation medium and to introduce a sample between the electrodes; anda second fluidic displacement system configured to cyclically displacethe separation medium and sample between a first direction generallyparallel to the direction of the electrodes and a second directiongenerally opposite the first direction.
 12. The apparatus of claim 11,wherein the top plate and bottom plate are stationary with respect tothe electrodes and each other.
 13. The apparatus of claim 11, whereinthe separation space between the top plate and bottom plate comprises athickness of about 0.01 to about 1.5 mm.
 14. The apparatus of claim 11,wherein the first fluidic displacement system comprises a multi-channelpump.
 15. The apparatus of claim 11, wherein the fluidic displacementsystem for introducing a separation medium and introducing a samplebetween the electrodes is further used to remove the sample afterseparation from the separation space.
 16. The apparatus of claim 11,wherein the second fluidic displacement system comprises a multi-channelpump.
 17. The apparatus of claim 11, further comprising a controllerconfigured to control the fluidic displacement systems.
 18. Theapparatus of claim 11, further comprising a controller configured tocontrol the flow of current between the electrodes.
 19. An apparatus forseparating particles comprising: an electrophoresis chamber defined by atop plate, a bottom plate and a plurality of electrodes generallyparallel to one another with a separation space disposed therebetween, afluidic displacement system configured to introduce a separation mediumand to introduce a sample between the electrodes and configured tocyclically displace the separation medium and sample between a firstdirection generally parallel to the direction of the electrodes and asecond direction generally opposite the first direction.
 20. Theapparatus of claim 19, wherein the top plate and bottom plate arestationary with respect to the electrodes and each other.
 21. Theapparatus of claim 19, wherein the fluidic displacement system comprisesa multi-channel pump.
 22. The apparatus of claim 19, further comprisingelectrode spacers configured to isolate the electrodes from theseparation space.
 23. The apparatus of claim 19, further comprising afraction collector outlet configured to displace the separated fractionsof the sample from the separation space into individual collectioncavities such as vessels or wells in microtiter plates.
 24. A method forseparating particles comprising: disposing a separation medium andsample in an electrophoresis chamber having a top plate, a bottom plate,a first chamber end, a second chamber end, and a plurality of electrodesgenerally parallel to one another with a separation space disposedtherebetween, the electrodes longitudinally extending toward each of theends, and a fluidic displacement system for conveying a separationmedium between the first and second chamber ends; applying a voltagebetween the electrodes effective to manipulate particleselectrophoretically; displacing at least a portion of the separationmedium and sample towards the first chamber end; and displacing at leasta portion of the separation medium and sample towards the second chamberend; and displacing at least a portion of the separation medium andsample towards the first chamber end.
 25. A computer executable softwarecode stored on a computer readable medium, the code for effectuating theseparation of particles in an electrophoresis chamber, theelectrophoresis chamber having a top plate, a bottom plate, and aplurality of electrodes generally parallel to one another with aseparation space disposed therebetween, and a fluidic displacementsystem for conveying a separation medium between the electrodes,comprising: code to enable application of a voltage between theelectrodes effective to manipulate particles electrophoretically; codeto enable displacement of at least a portion of the separation mediumand sample toward a first direction parallel to the direction of theelectrodes; code to enable displacement of at least a portion of theseparation medium and sample toward a second direction opposite thefirst direction; and code to enable displacement of the at least aportion of the separation medium and sample toward the first direction.